It is a standard procedure to prepare tissue samples for microscopic examination by embedding the tissue in paraffin and slicing the paraffin-embedded tissue very thinly with a microtome. Preparatory to embedding, the tissue is treated in various solutions appropriate to its examination and long-term stability. Typically, prior to paraffin embedding, the tissue sample is fixed, dehydrated, and cleared and then infiltrated with molten paraffin.
Typically, the tissue sample collected for examination is a unitary, connected portion of tissue, however, small parts of the tissue sample may be dislocated during tissue processing. Alternatively, a biopsy may be performed on minute fragments less than 1.0 mm in diameter, such as bronchial washings, cytology preparations and aspiration biopsies which may be gathered by skinny needles or imaging technology-guided tissue biopsy devises. Generally, this technique is called “tissue processing,” and it includes the following: (1) collection of the specimen; (2) fixation of the specimen to preserve tissue components; (3) sampling of a representative portion or aliquot; and (4) cutting of the non-processed tissue in a section plane to be presented to the microtome blade (for microscopic examination and information collection) and placement of this plane face down in a tissue processing cassette for containment during the “tissue processing phase.”
Information obtained in the microscopic study of biological tissue is taken from microtome cut sections that, on average, are less than 10 μthick. Thus, in view of the small size of the tissue sample and the precision with which the microtome cut sections are cut, it is of paramount importance to select a cutting plane and to maintain this plane as closely as possible, if not exactly, during processing, embedding and sectioning. Further, in the instance of minute specimens (approximately 1 mm or less), it may also be desirable to preserve the orientation of the tissue sample in relation to the adjacent structures from which it was collected. Such information may provide guidance for surgical or other treatments.
Tissue placed in a processing cassette may be fresh or fixed. The tissue sample is then passed through fixatives to remove water from the sample. Following dehydration, the tissue sample is then processed with a solvent that will dissolve fatty materials and “clear” the tissue sample. After being “cleared”, the tissue sample is placed in molten paraffin and it is infiltrated with the wax. Molten wax replaces the solvent which will evaporate or be diluted to trace levels, causing all the tissue to be infiltrated with a common wax binder.
Next, the cassette is opened and the infiltrated tissue is placed into a metal or similar base mold filled with melted wax. A considerable effort must be made at this moment to cause the “face down” selected cutting plane of the tissue to be positioned in the exact same planar relationship that was selected by the doctor or technician who placed the sample and selected the portion of the tissue that would be cut in microtomy. It is critical in many examinations, such as cancer diagnosis, to maintain the parallel relationships of: (a) selected cutting surface; (b) microtomy cut surface; (c) glass slide stained and covered cut section; and (d) microscopic section orientation for examination.
The technician thus must attempt to present the specimen to the microtome cutting blade in the exact position previously selected. The technician prepares the specimen for microscopic examination by subsequently mounting, staining and cover-slipping the microtome cut section on a microscope slide. Although it is desirable to maintain the positioning of the specimen, it is very difficult to control the position of the specimen particularly with minute tissue samples. A small (0.25-1.0 mm) specimen may likely shift its position in the cassette during processing or when it is removed from the processing cassette to the embedding mold prior to wax casting, so that the pre-selected position of the specimen may be lost.
Apparatuses are known that may be used for processing of tissue samples, such as described in U.S. Pat. Nos. 4,557,903 and 4,569,647, both to McCormick. In both the '903 and '647 patents, the tissue sample is deposited somewhat randomly in the cassette. It is a limitation that, although the tissue sample is disposed in an enclosed area, it may move freely within that area as the tissue sample is contacted with solvents during processing. Additionally, the apparatus of the '903 patent requires removing the tissue sample from the cassette and placing it in a mold for casting in paraffin. A shortcoming of the '903 and '647 patents is their inability to maintain the tissue sample in a desired orientation throughout processing.
There remains a need for an apparatus and processing method that permits the tissue plane selected by the surgeon or pathologist to be processed in situ without disturbing the orientation of the tissue specimen. More particularly, there is a need for an assembly and method that maintains the desired position of the tissue sample during the fixing, processing, and wax embedding steps, as well as during subsequent sectioning in a microtome and mounting of the desired section on a glass slide for staining, cover-slipping and microscopic examination. Such a system should be adaptable for both large specimen sections and minute fragments of less than 1 mm in size.